The present invention relates to the identification of microRNAs and mRNAs in in vitro culture media as non-invasive biomarkers of embryo development, and to methods of improving the viability of in vitro cultured embryos.
Infertility has become a challenge in many mammalian species. In humans, about 15% of couples fail to conceive within the course of a year of unprotected sex. Within the United States, 10.9% of women aged 15-44 have impaired fecundity and about 6% are infertile [1]. A decline in fertility over the last 40-50 years has also been observed in dairy cattle. Conception rates in the 1950s were reported to be about 55% for artificially inseminated cows at observed estrus and by 1990 the rate declined to about 35% [2]. Selection intensity for higher milk production in dairy cattle has propagated a paralleled decline in fertility, posing a challenge to dairy producers [3]. Across species, a culmination of factors contributes to the decline in fertility including parental genetic contribution and environmental factors.
Assisted reproductive technologies (ARTS) have become well developed and utilized to overcome the challenges of infertility. These technologies are costly and rather inefficient, as a 2012 Fertility Clinics Success Rates Report demonstrated that of all fresh non-donor ART cycles started in 2012, only 36% resulted in pregnancy and of those 29% resulted in live birth [4]. Furthermore, the pregnancy rate of cows following transfer of in vitro produced embryos is only 45% and have increased risk for abortion and still birth compared to cows with in vivo produced embryos [5]. Conventionally, assessment of embryo quality and potential of in vitro produced embryos, in both humans and cattle, is largely based on morphology [6]. Morphologically similar embryos, however, differ in developmental capacity likely due to differences in the underlying genetics driving development [7]. Thus, there is need for better, non-invasive methods and biomarkers for efficient selection of embryos with enhanced developmental potential that will result in a live birth.
MicroRNA (miRNA) biomarkers have recently been identified for detection of several pathologies in humans. Indeed, Mitchell et al. [8] demonstrated that miRNAs secreted from prostate cancer cells into the blood could be detected in a patient's serum sample, offering a non-invasive method for diagnosing cancer. The presence of miRNAs in the extracellular environment has recently been reported in many bodily fluids including serum, saliva, urine, and placental fluid [8-14]. Studies have demonstrated the extreme stability of miRNAs in serum as they withstand freezing, thawing and pH changes [8, 11]. In the extracellular environment, miRNAs are predominantly bound to proteins such as Ago2 and NPM1 which provides stability, though a fraction is contained within exosomes [9, 10, 12]. Secretion of miRNAs into the extracellular environment is cell-specific, allowing for the development of biomarkers for a wide range of cell types and tissues.
MiRNAs play a role in nearly every cellular process including cell proliferation, differentiation, metabolism, apoptosis and cell signaling [15]. These small non-coding RNA molecules act in cells to regulate gene expression. Mature miRNAs are 18-22 nucleotides in length and are found predominantly in the cytoplasm where they are assembled into RNA-induced silencing complexes (RISC) [16]. Once assembled within a RISC, the guide strand of the miRNA hinds to the 3′ UTR region of its target mRNA to either degrade or block translation of the mRNA, thereby blocking protein synthesis [16]. Imperfect binding to a miRNA's target mRNA allows for one miRNA to bind to a vast array of mRNA targets, increasing the regulatory potential of a single miRNA [17-19].
Embryonic development is partially driven by the underlying genetics of the embryo and as such miRNAs are dynamically expressed throughout the stages of development. In all cells, miRNA expression is precisely regulated in a developmental stage- and tissue-specific manner [18-20]. Similarly within gametes and embryos, the miRNA profile reflects differences in the competence and/or stage of development the cells are undergoing [21-24].
A study by Kropp et al. [25] provides evidence that miRNAs are differentially expressed between bovine blastocyst stage embryos and those which fail to develop, deemed degenerates. Therefore, miRNA expression in the gamete as well as the embryo is of great importance and dysregulation of key miRNAs could consequently alter development of the embryo.
Kropp et al. also reported the presence of miRNAs in bovine and human IVF culture media [25]. Similarly, Rosenbluth et al. [26] detected miRNAs in human IVF culture media and differential miRNA expression was correlated to pregnancy outcome and chromosomally abnormal embryos. These studies are limited in the number of miRNAs detected as they used only a few candidate miRNAs [22] or utilized a heterologous array for detection of miRNA [23]. Prior to the present invention, there has never been any report that the miRNA levels in the culture medium also were different among embryos of different developmental fate and can be used as indicators of embryo viability.
Likewise, mRNAs are packaged into exosomes and are secreted by cells into the extracellular environment (Valadi et al., 2007). However, there has been no report that specific mRNAs present in culture medium can be used as biomarkers for predicting embryo developmental prospect.
Furthermore, there has not been any report that supplementing or depleting specific miRNAs in the culture media can improve the developmental fate of an embryo.